Heterotrimeric G-Protein Shuttling Via Gip1 Extends the Dynamic Range of Eukaryotic Chemotaxis

Heterotrimeric G-protein shuttling via Gip1 extends the dynamic range of eukaryotic chemotaxis

Yoichiro Kamimuraa,b,1, Yukihiro Miyanagaa,b,1, and Masahiro Uedaa,b,2

aLaboratory for Cell Signaling Dynamics, Quantitative Biology Center (QBiC), RIKEN, Suita, Osaka, 565-0874, Japan; and bLaboratory for Single Molecular Biology, Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan

Edited by Peter N. Devreotes, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved March 7, 2016 (received for review September 11, 2015) Chemotactic eukaryote cells can sense chemical gradients over a adaptively over a wide range in the signal transduction cascades wide range of concentrations via heterotrimeric G-protein signaling; upstream of STEN. however, the underlying wide-range sensing mechanisms are only Insight into this question is provided by bacterial chemotaxis partially understood. Here we report that a novel regulator of G pro- and other sensory systems, such as photoreceptor rhodopsin (8). teins, G protein-interacting protein 1 (Gip1), is essential for extending Chemoreceptor methylation in bacteria confers a broad chemo- the chemotactic range of Dictyostelium cells. Genetic disruption of tactic range (11). In light adaptation, the phosphorylation of rho- Gip1 caused severe defects in gradient sensing and directed cell mi- dopsins in the visual system leads to rhodopsin down-regulation by gration at high but not low concentrations of chemoattractant. Also, arrestin, which blocks physical interaction with G-protein trans- Gip1 was found to bind and sequester G proteins in cytosolic pools. ducin (12). Phosphorylation-dependent receptor internalization is a Receptor activation induced G-protein translocation to the plasma feature of other systems for suppressing intracellular responses membrane from the cytosol in a Gip1-dependent manner, causing a (13). Overall, in these sensory systems, the chemical modifications biased redistribution of G protein on the membrane along a chemo- of receptors are important for regulating the dynamic range of the attractant gradient. These findings suggest that Gip1 regulates response. Consistently, Dictyostelium cells expressing unphosphory- G-protein shuttling between the cytosol and the membrane to ensure lated mutant cAR1 exhibit a narrow chemotactic range (14), and the availability and biased redistribution of G protein on the mem- phosphorylated cAR1s have reduced affinity for cAMP (15). Thus, brane for receptor-mediated chemotactic signaling. This mechanism chemical modifications of chemoattractant receptors are also im- offers an explanation for the wide-range sensing seen in eukaryotic portant in eukaryotic chemotaxis as a mechanism to extend the chemotaxis. chemotactic range. In addition to the receptor modifications, G proteins are phosphorylated and recruited from the cytosol to the eukaryotic chemotaxis | gradient sensing | dynamic range extension | plasma membrane upon receptor stimulation in Dictyostelium cells heterotrimeric G protein (16, 17), although the relevance of these actions on wide-range sensing and adaptation is unknown. hemotaxis in eukaryotic cells is observed in many physio- Here we report that a novel regulator of G proteins, G protein- Clogical processes including embryogenesis, neuronal wiring, interacting protein 1 (Gip1), is essential for the wide-range che- wound healing, and immune responses (1, 2). Chemotactic cells motaxis in Dictyostelium cells. Gip1 regulates G-protein localiza- share basic properties including high sensitivity to shallow gradients tion between the cytosol and plasma membrane upon receptor and responsiveness to a wide dynamic range of chemoattractants activation, which targets cytosolic G proteins to the membrane in (3, 4). For instance, human neutrophils and Dictyostelium cells can a biased manner along chemoattractant gradients at higher sense spatial differences in chemoattractant concentration across chemotactic ranges. These findings provide evidence for a wide- the cell body in shallow gradients as low as 2% and exhibit che- range sensing mechanism in which Gip1-dependent G-protein motaxisovera105–106-fold range of background concentrations – (5 7). Thus, wide-range sensing and adaptation are critical features Significance of chemotaxis as well as other sensory systems such as visual signal transduction (8). However, the underlying regulatory mechanisms Eukaryotic chemotactic cells can recognize chemical gradients in eukaryotic chemotaxis remain unclear. over a wide range of concentrations. This ability is physiolog- The molecular mechanisms of chemotaxis are evolutionarily ically important for numerous biological processes; however, conserved among many eukaryotes that use G protein-coupled its underlying mechanism is unknown. Here we report that the receptors (GPCRs) and heterotrimeric G proteins to detect dynamic range of chemotaxis is extended to higher concen- chemoattractant gradients (3, 4). In Dictyostelium cells, extracellular trations by gradient sensing achieved via regulation of trimeric cAMP works as a chemoattractant, and binding to its receptor cyclic α βγ G-protein shuttling between the cytosol and plasma mem- AMP receptor 1 (cAR1) activates G proteins (G 2G ) along the brane. G protein-interacting protein 1 (Gip1) regulates this in- concentration gradient, leading to the activation of multiple sig- tracellular G-protein translocation, which redistributes cytosolic – – – naling cascades including the PI3K PTEN, TorC2 PDK PKB, G proteins to the plasma membrane along chemical gradients at phospholipase A2, and guanylyl cyclase pathways. In contrast to the high chemoattractant concentrations. This dynamic spatiotemporal spatial distributions of cAMP/cAR1 association and G-protein ac- regulation of trimeric G protein yields proper processing of tivation, downstream signaling pathways are activated in an ex- receptor-mediated signaling. tremely biased manner at the anterior or posterior of the cell (3, 4). For example, localized patches of phosphatidylinositol 3,4,5- Author contributions: Y.K., Y.M., and M.U. designed research; Y.K. and Y.M. performed research; Y.K. and Y.M. analyzed data; and Y.K., Y.M., and M.U. wrote the paper. trisphosphate (PIP3) are generated at the plasma membrane by an intracellular signal transduction excitable network (STEN) and The authors declare no conflict of interest. function as a cue to control the pseudopod formation of motile cells This article is a PNAS Direct Submission. (9,10).BecausePIP3 patches have a relatively constant size of a few 1Y.K. and Y.M. contributed equally to this work. microns in diameter, this excitable mechanism can ensure a con- 2To whom correspondence should be addressed. Email: [email protected]. stant output of chemotactic responses over a wide range of concen- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. trations. However, it is unclear how chemical gradients are sensed 1073/pnas.1516767113/-/DCSupplemental.

4356–4361 | PNAS | April 19, 2016 | vol. 113 | no. 16 www.pnas.org/cgi/doi/10.1073/pnas.1516767113 Downloaded by guest on September 23, 2021 translocation ensures the availability and biased redistribution of The interaction between Gip1 and Gβ was verified by a pull- G protein for receptor-mediated signaling at the higher range, down assay using GFP-Flag–tagged Gip1 (Gip1-GFPF), as shown which is in contrast to the chemical modification mechanisms in Fig. 1B. To identify the interaction region, the N-terminal PH underlying adaptation by sensory receptors. domain (amino acids 1–109) and the Gip1 C terminus (amino acids 108–310) were separately expressed and used in the assay. The C Results but not N terminus bound to Gβ as efficiently as the full-length Gip1 Is an Interactor of Trimeric G Protein. We identified Gip1 by protein (Fig. 1C). Furthermore, to determine whether G proteins using a tandem affinity purification (TAP) tag of Gβ.FortheTAP bind Gip1, the proteins that copurified with Gip1-GFPF by TAP assay, whole-cell extracts (WCEs) were prepared from cells in were analyzed by MS (Fig. S1 D and E). The two prominent bands β α vegetative growth and chemotactically competent cells with or at 35 and 40 kDa in Fig. S1D were G and G subtypes, re- spectively, including Gα4, Gα9, Gα5, and Gα2. Together with the without cAMP stimulation. Under these conditions, four bands – were observed at around 240, 40, 30, and 9 kDa in addition to the additional data in SI Text (Fig. S1 F K), these results confirmed that Gip1 binds to G proteins via the C terminus. TAP-tagged Gβ protein (Fig. 1A and Fig. S1A). Mass spectrometry The physiological roles of Gip1 in the development of Dictyos- (MS) analysis revealed that p40 and p9 were Gα4andGγ,re- β telium cells were investigated by examining the phenotype of gip1- spectively; p240 was ElmoE, which is a known G -binding protein knockout (gip1Δ) cells (Fig. S1L). Wild-type (WT) cells formed (18); and p30 was a previously uncharacterized protein encoded by streams that consisted of a collective migration of chemotactic cells DDB_G0271086 and designated as Gip1. Gip1 contained a pleckstrin upon starvation, whereas gip1Δ cells caused smaller aggregates with homology (PH) domain at the N terminus between amino acids reduced stream formation (Fig. 1D). Moreover, Gip1 over- 1–109 and an unidentified region at the C terminus. Gip1 homo- expression in WT cells (Gip1OE cells) delayed progression of the logs exist in other protists, such as Entamoeba and Acanthamoeba early development, suggesting requirements to maintain the WT (Fig. S1 B and C). In addition, the Gip1 C terminus, where there is expression level for normal development (Fig. S1M). The early no similarity to known G-protein interactors including the regu- developmental defects in gip1Δ cells were rescued by ectopically lator of G proteins signaling, has weak but significant similarity to expressing Gip1-GFPF but not the N or C terminus (Fig. 1D), in- tumor necrosis factor α-induced protein 8 (TNFAIP8) in Homo dicating that normal development depended on the full length of sapiens (Fig. S1 B and C) (19). Gip1. gip1Δ cells expressed normal levels of endogenous cAR1, which is a marker of transcriptional regulation in early develop- ment, indicating normal transcriptional regulation in gip1Δ cells but defects in cell aggregation (Fig. S1N). TAP: F2G-Gβ A B wt cells WCE IP: α-Flag v 0” 30” Gip1 Is Essential for Chemotaxis at Higher Concentration Ranges. none + + Gip1-GFP-Flag Because chemotaxis is a major event for cell aggregation, we + + Δ Gα4-GFP-Flag + + examined the chemotactic ability of gip1 cells. Unusual phe- kDa notypes of gip1Δ cells showed defects in the chemotactic range. Gβ

cAMP was applied in a gradient with a micropipette such that CELL BIOLOGY 250 ElmoE Gα4-GFPF the concentration decreased with distance from the tip (Fig. S2A, 150 Gip1-GFPF Upper Right). At 100 μM cAMP in the micropipette, WT cells displayed efficient chemotaxis independent of the distance from 100 C the micropipette tip and ultimately reached the tip (Fig. 2A and 75 gip1Δ cells WCE IP: α-Flag F2G-Gβ Movie S1). gip1Δ cells showed efficient chemotaxis at lower Gip1(Full)-GFPF + + cAMP concentration regions, but severe defects were observed 50 Gip1(C)-GFPF + + at higher concentration regions near the tip, resulting in an area Gα Gip1(N)-GFPF 37 + + of exclusion there (Fig. 2A and Movie S2). The chemotactic in- Gip1 Gβ dex (CI), which was calculated based on distance from the mi- 25 cropipette tip, increased for WT cells but decreased for gip1Δ 20 Gip1(Full)-GFPF cells at higher cAMP concentrations (Fig. 2B, Upper Left panels). Δ 15 Gip1(C)-GFPF gip1 cells had slightly reduced motility compared with WT cells μ 10 Gγ Gip1(N)-GFPF that had similar dependence on distance from the tip. At 10 M cAMP in the micropipette, however, gip1Δ cells exhibited effi- D gip1Δ cient chemotaxis. These chemotactic abilities were confirmed by OE wt gip1Δ Gip1 +Gip1-GFPF +Gip1(N)-GFPF +Gip1(C)-GFPF the small population assay (see Fig. S2B for the method) (20). 9 h 9 h 9 h That is, WT cells exhibited efficient chemotaxis in response to 0.1–100 μM cAMP, whereas gip1Δ cells exhibited reduced che- motaxis at concentrations >10 μM, thus showing a narrower chemotactic range (Fig. 2C and Fig. S2C). These chemotactic defects of gip1Δ cells at higher cAMP concentrations were res- Fig. 1. Gip1 is an interactor of trimeric G protein. (A) TAP using Flag×2-GFP- cued by the ectopic expression of full-length Gip1, but not of the – Gβ (F2G-Gβ) in vegetative growth (v) and before (0’’) and 30 s after (30’’) Gip1 N or C terminus (Fig. S2A and Movies S3 S5). OE Δ cAMP stimulation. Silver staining showed F2G-Gβ copurified with Gip1 along Gip1 and gip1 cells had distinct effects on the chemotactic with ElmoE, Gα, and Gγ.(B) Gip1-GFP-Flag (Gip1-GFPF) and Gα4-GFP-Flag ranges (Fig. 2C). Gip1OE cells had severely impaired chemotaxis (Gα4-GFPF) proteins were expressed in WT cells and immunoprecipitated (IP) at lower cAMP concentrations but exhibited normal chemotaxis with α-Flag antibody from WCE. Copurification of Gβ (Upper) and GFP-Flag at higher concentrations. Overexpression of the Gip1 C terminus α (Lower) was evaluated by immunoblotting. G 4-GFPF was used as a positive also impaired chemotaxis, but differently, as chemotaxis was control and confirmed the coprecipitation of Gβ.(C) Full-length Gip1 and its C and N termini tagged with GFP-Flag (Gip1-GFPF) were immunoprecipitated inhibited at the lower and higher concentration ranges but pre- with α-Flag antibody from WCE. Copurification of Gβ (Upper) and GFP-Flag served at concentrations in between (Fig. 2C). These results (Lower) was evaluated by immunoblotting. (D) Cells were starved on non- suggest that full-length Gip1 at a normal expression level is re- nutrient DB agar. Gip1OE cells are WT cells with full-length Gip1 overexpression. quired for wide-range chemotaxis.

Kamimura et al. PNAS | April 19, 2016 | vol. 113 | no. 16 | 4357 Downloaded by guest on September 23, 2021 A wt 0’ 60’ gip1Δ 0’ 150’

Fig. 2. Chemotactic dynamic range is extended to higher concentrations via Gip1. (A) Chemical gradi- ents were applied to WT and gip1Δ cells from a micropipettetipfilledwith100μM cAMP. Repre- 1.0 20 sentative images of cell trajectories (red lines) are B 100 μM cAMP wt 100 μM cAMP C 15 gip1Δ 1.0 wt shown before (0’) and 60 or 150 min after (60’ and 0.5 10 gip1Δ 150’, respectively) the start of the assay. Cells are OE 5 Gip1 highlighted in green. (Scale bar, 50 μm.) (B)CIand

amined droplet) Gip1(C) motility speed were calculated from the assay in A

0.0 0 x

0 150 300 450 750600 0 150 300 450 750600 e 0.5 /gip1Δ μ 1.0 20 / following application of 10 or 100 M cAMP at dif- 10 μM cAMP 10 μM cAMP 15 ferent distances from the micropipette tip. Each bar Response oplet r represents the average of at least 10 cells. (C)The 0.5 10 d chemotactic response to different cAMP concentra- Chemotactic index

5 ded 0.0 Motility speed (μm/min) n tions was evaluated by the small population assay 0.0 0 0 150 300 450 750600 0 150 300 450600 750 -8 -7 -6 -5 -4 -3 -2 (Fig. S2B). Data represent the mean ± SEM of three espo r Distance from pipette (μm) ( log [cAMP] experiments.

Uniform Receptor Stimulation Induced Normal Signaling Responses in Gip1 Regulates G-Protein Shuttling upon Receptor Stimulation. Be- gip1Δ Cells. To identify the defects in the chemotactic signaling of cause chemotaxis-competent cells redirect G proteins from the gip1Δ cells, we followed the chemoattractant-triggered signaling cytosol to the plasma membrane upon cAMP stimulation (17), we reactions (3, 4). Cells were uniformly stimulated with cAMP, and investigated whether Gip1 is involved in this process. In WT cells, the phosphorylation of cAR1, Gα2, PKBR1, PKBA, and Erk2 as Gα2 was clearly recruited to the membrane upon cAMP addition, well as fluorescence resonance energy transfer (FRET) changes which was assessed by the decreased levels of cytosolic Gα2-TMR OE between G-protein subunits, PIP3 production at the plasma (Fig. 4 A and B,andMovie S6). Gip1 cells showed similar membrane, and actin polymerization were evaluated (Fig. S3). As cAMP-induced Gα2 translocation, with a half-maximum at around describedindetailinSI Text, no obvious differences in these sig- 10 nM cAMP (Fig. 4 A–C). However, the same was not true of naling reactions upon uniform cAMP stimulations were observed gip1Δ cells even though there was less Gα2-TMR protein in the in gip1Δ or WT cells, showing that the uniform increase of cAMP cytosol under resting conditions (Fig. 4 A–C). Instead, the amounts from the basal level can induce normal activation of chemotactic of Gα2 on the membrane decreased slightly with the cAMP con- signaling pathways in gip1Δ cells. Thus, cells can detect the initial centration in gip1Δ cells (Fig. 4 B and C). Biochemical fraction- increase of cAMP concentrations in a Gip1-independent manner. ation analysis of Gα2 localization confirmed these observations (Fig. 4D). Additionally, unlike G protein, Gip1 remained in the Gip1 Sequesters Trimeric G Protein in Cytosolic Pools. We next in- cytosol upon cAMP addition (Fig. S4 E and F). Together with the vestigated the subcellular localization of G proteins, given that detailed characterization described in SI Text, we concluded that they are modified upon cAMP stimulation (17). Gα2andGγ cAMP-induced Gα2 translocation depends on Gip1 but not on Ras α conjugated with the fluorescent dye TMR (G 2-TMR and TMR- activation, PIP3 production, or actin polymerization (Fig. S5). Gγ, respectively) via a Halo tag were used for localization analysis. Next we sought to identify the region of Gip1 required for Gα2-TMR and TMR-Gγ were present in both the cytosol and G-protein translocation. Full-length Gip1 expression in gip1Δ cells plasma membrane of WT cells, whereas the cytosolic fractions of rescued Gα2-TMR translocation upon cAMP stimulation (Fig. 4 A both proteins were significantly decreased in gip1Δ cells (Fig. 3A and B and Movie S6). The Gip1 N terminus did not revert Gα2to and Fig. S4A). Gip1OE cells exhibited an increase in the cytosolic the resting-state cytosolic localization, nor did it rescue cAMP- fraction of G proteins, which was also observed by expression of induced Gα2 translocation (Fig. 4 A and B). In contrast, the C the Gip1 C terminus but not of the Gip1 N terminus. These ob- terminus caused cytosolic retention of Gα2, an effect that was servations were confirmed for endogenous Gα2 and Gβ by bio- unaltered by cAMP stimulation (Fig. 4 A–C). The C terminus is chemical fractionation into cytosolic and membrane components therefore sufficient for tethering G proteins in the cytosol but (Fig. 3B). The cytosolic amounts of Gα2andGβ were significantly insufficient for inducing G-protein translocation to the mem- reduced to about 20% and 40%, respectively, in gip1Δ compared brane upon cAMP stimulation, suggesting regulatory roles of the with WT cells. Overexpression of the Gip1 and Gip1 C terminus N-terminal PH domain in G-protein translocation. Sequestration of compared with vector control increased cytosolic Gα2 by 2.3-fold cytosolic G protein via Gip1 C terminus is inhibitory for chemotaxis and 1.6-fold, respectively, and cytosolic Gβ by 3.8-fold and 2.0- because the proteins available for receptor-mediated signaling are fold. The lesser effect of Gip1 C terminus compared with full- depleted from the membrane. Upon receptor stimulations at the length Gip1 is attributed to a difference in the expression level of higher concentration ranges, G protein translocates to the mem- these proteins (Fig. S4B). Thus, the cytosolic localization of G brane, probably by releasing from Gip1, thus serving as a possible proteins requires Gip1, likely by complex formation via its C ter- receptor-mediated signal (see Discussion for details). minus in the cytosol (Fig. 1C). Consistently, the full-length and N and C termini of Gip1 were localized mostly in the cytosol (Fig. gip1Δ Cells Fail to Process Gradient Signals at Higher Ranges. As S4C). The biochemical fractionation assay confirmed that en- shown in Fig. 2, gip1Δ cells exhibited decreased chemotaxis with- dogenous Gip1 as well as ectopically expressed full-length and the out severe motility defects, suggesting that Gip1-dependent N and C termini of Gip1 were predominantly found in the G-protein translocation is involved in gradient sensing. To estimate supernatant fraction (Fig. S4 C and D). Thus, Gip1 is responsible theextentofgradientsensing,PIP3 production was evaluated by using for maintaining the cytosolic pools of G proteins in the absence the specific probe PHAKT-GFP (21, 22). When 10 μMcAMPwas of chemoattractants. applied as a gradient by a micropipette to WT cells, a crescent-like

4358 | www.pnas.org/cgi/doi/10.1073/pnas.1516767113 Kamimura et al. Downloaded by guest on September 23, 2021 OE Δ A wt gip1Δ Gip1 gip1 cells (Fig. 5B), which is consistent with the kinetics of PIP3 production upon uniform stimuli (Fig. S3C). Differences between WT and gip1Δ cells were observed after the initial responses, the so- called second response. The restricted accumulation of PHAKT-

G α 2-TMR GFP was induced at the high side of the gradient in WT cells showing gradient sensing but not in gip1Δ cells (Fig. 5B). The di- minished signals were never retained in gip1Δ cells. These data indicate that gip1Δ cells can detect information about the initial increase in cAMP but not gradient information with sufficient ac- curacy at higher chemoattractant ranges. Gip1(full) / gip1Δ Gip1(N) / gip1Δ Gip1(C) / gip1Δ We further assessed Gip1-dependent Gα2 translocation under cAMP gradients by using a theta glass pipette with two separate chambers containing 0 and 10 nM cAMP solutions, respectively (Fig. 5C, Top Left). In WT cells, Gα2-TMR accumulated in the

G α 2-TMR TMR-G γ area stimulated with a higher cAMP concentration (Fig. 5C, arrowheads). The fluorescence intensities of Gα2-TMR in the stimulated region were 1.4-fold higher than in the unstimulated region. In contrast, the uniform distribution of the fluorescence signals along the entire membrane was not changed in gip1Δ cells TMR-G γ upon gradient stimulation (Fig. 5C). That is, receptor stimulation α B wt induced G 2 accumulation at the stimulated region of the wt gip1Δ empty vector Gip1(full) Gip1(N) Gip1(C) membrane in a Gip1-dependent manner. Thus, Gip1-dependent WS PWS P WSPWSPWSPWSP G-protein translocation can underlie the availability and biased Gα2 redistribution of G protein at the membrane under chemical gradients at the higher range. Moreover, the full availability of G Gβ protein to the membrane is insufficient for efficient gradient Gα2GGβ Gα2 β p=0.35 p=0.61 1.0 1.0 p=0.42 * 0.8 *** * 0.8 *** ** Gα2-TMR 0.6 0.6 A OE Gip1(full) Gip1(N) Gip1(C) 0.4 0.4 wt gip1Δ Gip1 / gip1Δ / gip1Δ / gip1Δ 0.2 0.2

relative amount 0.0 0.0 Gip1(C) Gip1(full)Gip1(N)Gip1(C) wt gip1Δ wt gip1Δ empty Gip1(full)Gip1(N) empty of cytosolic G protein vector vector - cAMP CELL BIOLOGY

Fig. 3. Gip1 regulates shuttling of trimeric G protein between the cytosol and the plasma membrane. (A) Intracellular distribution of Gα2-TMR and TMR-Gγ, respectively. Gip1(full), Gip1(N), and Gip1(C) represent the coexpression of the full-length (full), N terminus, and C terminus of Gip1, respectively. (Scale bar, + cAMP μ 10 m.) (B) WCEs (W) prepared from the indicated cell lines were separated wt Gip1(N) / gip1Δ 1.1 B 1.4 1.4 C into supernatant (S) and pellet (P) fractions. Endogenous Gα2andGβ were gip1Δ Gip1(C) / gip1Δ OE detected by immunoblotting (Top). The fraction of the supernatant was calcu- 1.3 Gip1 1.3 Gip1(full) / gip1Δ < < < cAMP stimulation cAMP stimulation lated by band intensities (Bottom). *P 0.05, **P 0.01, ***P 0.005, t test. cytosol 1.2 1.2

(10 μM) (10 μM) ) n i

P 1.0 1.1 1.1 cAM - accumulation of PHAKT-GFP was observed in areas close to 1.0 1.0 rmalized) wt o

higher cAMP concentrations (Fig. 5A, Fig. S6A, Upper Left,and n 0.9 0.9 0.9 ( gip1Δ Δ (+cAMP/ Movie S7), whereas gip1 cells did not exhibit the same bias in OE 0.8 0.8 Gip1 PH -GFP localization under cAMP gradients with high back- AKT 0.7 0.7 Gip1(C) / gip1Δ Fluorescence intensity in cytosol ground concentrations (Fig. 5A, Fig. S6A, Upper Right,andMovie Fluorescence intensity 0.8 50 050100-50 050100 0 -10-9 -8 -7 -5-6 -4 S8). In lower cAMP concentrations supplied by a micropipette D Time (sec) Time (sec) log [cAMP] containing 0.1 μM cAMP, crescent-like distributions of PH - wt gip1Δ wt gip1Δ AKT Time 1.0 GFP near the source were observed in WT cells and also in gip1Δ (min) 0 0.5 2 0 0.5 2 WSPWSP WSP WSPWSPWSP cells but with less efficiency (Fig. 5A and Fig. S6A, Lower panels). 0.5

Gα2 of sup To further confirm the defects in gradient sensing of gip1Δ cells, we 0 0’ 0.5’ 2’

used Ras binding domain (RBD)-GFP to detect activated Ras, Relative intensity Time (min) which is another reporter for the cAMP signaling pathway (4). Δ Fig. 4. Gip1 modulates spatial regulation of trimeric G protein in response to RBD-GFP behaved similarly to PHAKT-GFP in WT and gip1 cells chemoattractants. (A) Fluorescent images of Gα2-TMR. Cells were treated with (Fig. S6 B and C). These results indicate that Gip1 is required for cAMP in the presence of 5 μM latrunculin A. (Scale bar, 10 μm.) (B)Cells gradient sensing at higher chemoattractant concentrations. expressing Gα2-TMR were stimulated with cAMP, and fluorescence intensity in ± We next examined the spatiotemporal dynamics in PHAKT-GFP the cytosol (mean SD) was quantified at 4-s intervals. (C) Translocation of localization upon gradient stimulation. Gradient stimulation ini- Gα2-TMR to the plasma membrane was assessed quantitatively by the de- tially caused a uniform increase in PH -GFP signals along crease in cytosolic fluorescence intensity. Gα2-TMR protein was expressed in AKT Δ the entire contour of WT cells within 10 s, and this effect was WT and gip1 cells harboring Gip1-GFPF (full-length and C terminus). A dose- dependent curve was plotted (mean ± SD). (D) Cells were fractionated before followed by an abrupt signal reduction on the membrane (Fig. 5B, and after stimulation with 1 μMcAMPfor0.5and2min(Left). The signal Upper panels), which is due to the excitable property of PIP3 pro- intensity of the supernatant fraction relative to that of WT cells at 0 min was duction (10, 23, 24). These initial responses were similar in WT and quantified (Right).

Kamimura et al. PNAS | April 19, 2016 | vol. 113 | no. 16 | 4359 Downloaded by guest on September 23, 2021 yt -10 0” 10” 30” 120” isnet wt A Source of cAMP B C 1.5 P<0.005

) gip1Δ

b -20 ni 0 0 no P=0.36 20 ecnecseroul b cAMP iger/a cAMP-filled pipette 1.0 10 Perimeter index

wt noiger 3 a 10 nnMM

fevi 0.5 cAMPM

( lized)

tale 2.0 2.0 a 2 wt 10 μM cAMP wt 0.1 μM cAMP

R 0.0

1.5 1.5 (norm 1 localno no local wt / no stimulus wt / local stimulus Fluorescence intensity 1.0 1.0 -20 0 20 40 60 80 100 120 Time (sec) 0.5 0.5 0” 10” 30” 120”

0.0 0.0 2.0 2.0 gip1Δ 10 μM cAMP gip1Δ 0.1 μM cAMP 1.5 1.5 closer region gip1Δ / no stimulus gip1Δ / local stimulus 3 gip1Δ far region 1.0 1.0

Fluorescence intensity (normalized) 2 0.5 0.5

(normalized) 1 0.0 0.0 Fluorescence intensity -20 -10 0 10 20 -20 -10 0 10 20 -20 0 20 40 60 80 100 120 Perimeter index (a.u.) Time (sec)

Fig. 5. Gip1 is involved in the signal transduction of chemical gradients. (A) Gradient sensing monitored by a PIP3 probe, PHAKT-GFP. Cells were treated with 5 μM latrunculin A and stimulated with a micropipette tip containing 10 or 0.1 μM cAMP. The cell perimeter was divided into 40 points (Top); relative in-

tensities (mean ± SD) of PHAKT-GFP in WT and gip1Δ cells are shown as histograms in green and red, respectively. (B) Representative temporal changes of PHAKT-GFP localization in WT (Top) and gip1Δ (Bottom) cells when stimulated by a micropipette containing 10 μM cAMP. Red arrows show the direction of the gradient source. Fluorescence intensities at the near (orange diamond) or far (blue circle) side of a cell from the micropipette were plotted over timeasthe mean ± SD. (Scale bar, 10 μm.) (C)Gα2 localization under steep cAMP gradients. Cells expressing Gα2-TMR were stimulated by a theta micropipette with two separate chambers containing either 0 or 10 nM cAMP (Top Left), and Gα2-TMR localization was visualized (Bottom). The left and right images show pre- and poststimulation, respectively. Ratios of the fluorescence intensities in the regions a and b were measured (Top Right, mean ± SD). (Scale bar, 10 μm.)

sensing at the higher range, as seen by gip1Δ cells lacking the Based on these observations, we propose a model in which the role translocation-mediated redistribution. of Gip1 in G-protein translocation for wide-range chemotaxis can be Such defects in gradient sensing by gip1Δ cells were also ob- explained by the following three steps (Fig. S7). First, Gip1 se- served in moving cells. F-actin, as monitored by LimEΔcoil-RFP questers the cytosolic pools of G proteins through binding at the C (25), was selectively localized at the higher cAMP gradient in terminus, where cytosolic G proteins are not available for chemo- WT cells, but the region of this signal was weakly restricted in tactic signaling on the membrane (Figs. 1 and 3). Second, receptor gip1Δ cells (Fig. S6D). Similarly, RBD-GFP and PHAKT-GFP stimulation facilitates G-protein translocation from the cytosol to were localized much more broadly in gip1Δ than in WT cells (Fig. the membrane depending on the N-terminal PH domain of Gip1 S6D). These data suggest that Gip1 confines pseudopod forma- (Fig. 4). Third, Gip1-dependent translocation supplies a signal tion along chemical gradients through the regulation of G-protein transducer for chemoattractant receptorsathigherrangesand translocation for efficient chemotaxis. causes a biased redistribution of G proteins on the membrane along chemical gradients (Fig. 5). This biased redistribution contributes to Discussion proper gradient sensing at higher concentration ranges. Identification of Gip1 as a Novel Regulator of Trimeric G Protein. Chemotaxis ranges observed in the cells with the knockout, Using TAP of Gβ, we identified four interacting proteins with high overproduction, and truncation of Gip1 can be explained by specificity: Gα,Gγ, ElmoE, and Gip1 (Fig. 1A). ElmoE associates G-protein dynamics in G-protein subcellular localizations—that is, with Gβγ and serves as a guanine nucleotide exchange factor for sequestering in the cytosol, translocation to the membrane, and Rac to control F-actin in pseudopods (18). Because the interaction biased redistribution on the membrane. Because G protein on between G proteins and ElmoE was unaffected in gip1Δ cells (Fig. the membrane is responsible for chemotactic signaling, its cyto- S1D), Gip1 and ElmoE function independently. We found Gip1 solic sequestering by Gip1 inhibits chemotaxis. G-protein trans- can bind to various Gα subtypes, such as Gα2, Gα4, Gα5, and Gα9 location upon receptor stimulation can eliminate the inhibitory (Fig. S1 D and E), although phenotypic defects of gip1Δ cells were effects of Gip1, resulting in fully available G protein on the obvious only in Gα2-dependent chemotaxis in response to cAMP membrane. Gip1OE cells have impaired chemtotaxis at the lower during early development. As discussed below, our analyses sug- but not higher ranges because of excessive sequestration but gest Gip1 functions as a regulator of G-protein localization be- functional translocation of cytosolic G protein. On the other tween the cytosol and the plasma membrane. hand, C-terminal Gip1OE cells have impaired chemotaxis at the lower and higher ranges because C-terminal Gip1 loses the Gip1-Dependent G-Protein Translocation for Wide-Range Chemtotaxis. translocation activity of G protein but still maintains sequestra- Previous studies have revealed that G proteins undergo trans- tion activity. In the case of gip1Δ cells, G protein is fully available location to the membrane upon receptor stimulation (17). We found over a wide range but has an impaired biased redistribution of G that Gip1 regulates G-protein translocation by its sequestering protein, leading to chemotactic defects at the higher ranges. Thus, function (Figs. 3 and 4) and is required for chemotaxis at higher it is likely that the proper regulation of G-protein availability by ranges (Figs. 2 and 5). Furthermore, we found that receptor stim- Gip1 is a prerequisite for gradient sensing over a wide range. ulation induced a biased redistribution of G protein under chemical Recently, it was reported that the Ric8 homolog in Dictyostelium gradients on the membrane in a Gip1-dependent manner (Fig. 5). cells contributes to chemotaxis by amplifying G-protein activity at

4360 | www.pnas.org/cgi/doi/10.1073/pnas.1516767113 Kamimura et al. Downloaded by guest on September 23, 2021 lower cAMP concentrations (26). Thus, the wide sensitivity to Materials and Methods chemical gradients could be achieved by multiple mechanisms, Chemotaxis Assay. Before the assay, cells were treated to proceed with their including receptor phosphorylation, Ric8, and Gip1. development as previously described (20). For the small population assay, ∼3,000 developed cells in a 1-μL droplet on the nonnutrient agar plate was placed next Gip1-Dependent Gradient Sensing. We show evidence that Gip1 is to a drop of cAMP solution. The distance between the centers of the two droplets was 2 mm. After 30 min, a responded droplet was assessed by cell ac- involved in gradient sensing at higher chemotactic ranges (Fig. 5). × 5 Δ cumulation at the edge near a cAMP droplet. For the micropipette assay, 2 10 Under uniform chemoattractant concentrations, gip1 cells exhibited developed cells were seeded on a glass-bottom dish, and cAMP gradients were receptor-induced reactions almost normally (Fig. S3). Upon gradient applied by a Femtotip microcapillary (Eppendorf) containing 10 or 100 μMcAMP – stimulations, gip1Δ cells initially distributed PIP3 along the entire and ATTO 532 (AD 532 21; ATTO-TEC GmbH) with a constant pressure of 10 hPa. plasma membrane (Fig. 5B). These results indicate that a sudden increase of receptor stimulation can be transduced by G protein Gradient Sensing Assay by PHAKT-GFP Measurements. Cells expressing PHAKT- without Gip1 leading to the excitation of PIP , suggesting that STEN GFP were subjected to cAMP gradients by a Femtotip microcapillary containing 3 100 nM or 10 μM cAMP in the presence of 5 μM latrunculin A. Fluorescence works normally without Gip1. This excitable reaction was sub- images of PHAKT-GFP were acquired by confocal microscopy at 1- or 10-s in- sequently followed by the biased accumulation of PIP3 along chem- tervals. Fluorescence intensities were measured along the plasma membrane of ical gradients in WT cells, whereas this event was not observed in each cell and then normalized by the average intensity of the perimeter. gip1Δ cells (Fig. 5 A and B). During the biased PIP3 accumulation, α cells obtained gradient signals by adapting to the average concen- Trimeric G-Protein Translocation. Starved cells expressing G 2-TMR and TMR- Gγ were treated with 5 μM latrunculin A. Cells were stimulated uniformly by tration of the chemoattractant across the cell body, which can be adding 200 μL of various concentrations of cAMP solution to 10 μL of a cell explained by a local excitation and global inhibition (LEGI) model suspension droplet in a glass-bottom dish. Fluorescence images were acquired combined with STEN (LEGI–STEN model) (10). LEGI works as an by confocal microscopy at 4-s intervals, and the intensities on the plasma adaptive network that detects gradient signals of various concentra- membrane and the cytosol were measured. tions by combining the excitor and the inhibitor, which reflect the local and the average concentration of the chemical gradient, re- Cell Preparations and Other Methods. Dictyostelium discoideum cells were used for all experiments. Full methods and any associated references are spectively. STEN works as an amplifier of the LEGI output to gen- described in SI Materials and Methods. erate a localized signal in an all-or-none fashion. According to the LEGI–STEN model, gip1Δ cells likely have defective LEGI but ACKNOWLEDGMENTS. We thank S. Taguchi and K. Tanabe for technical assistance; T. Miyagawa for purified Gip1 proteins; and Y.K., Y.M., and M.U. functional STEN, because the initial PIP3 excitation was normal but laboratory members for helpful discussion and advice. We thank Dr. P. Devreotes the biased PIP3 accumulation was not. Our observations also suggest and his laboratory members for helpful discussion and advice. We are also grateful that the Gip1-dependent translocation and redistribution of G pro- to Dr. R. Nakagawa for MS analysis and P. Karagiannis for reading the manuscript. – – tein on the membrane can contribute to a gradual localization of rasC and rasG cells were provided by National BioResource Project (NBRP)-Nenkin. We appreciate the basic information from the DictyBase. This work was supported excitor in chemotactic signaling at higher chemotactic ranges, which by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant 24570224 expands the dynamic range for chemotaxis. (to Y.K.). CELL BIOLOGY 1. 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